HDA法用于检测肉中金黄色葡萄球菌的研究

Detection of Staphyloccocus aureus in Meat by Helicase-dependent Isothermal DNA Amplification Assay

摘要:

本文研究了猪肉、牛肉、羊肉、鸡肉中金黄色葡萄球菌的依赖解旋酶恒温基因扩增检测方法(HDA法)，以金黄色葡萄球菌耐热核酸酶nuc基因为目的基因，设计一对特异性引物，优化反应条件UvrD helicase、T4 gp32的浓度，通过赖解旋酶恒温基因扩增方法直接检测猪肉、牛肉、羊肉、鸡肉中金黄色葡萄球菌，扩增产物通过电泳进行检测。结果表明，HDA法检测肉中金黄色葡萄球菌的特异性强，试验涉及的其它菌株未发生扩增，只有金黄色葡萄球菌得到与设计序列长度一致的216 bp基因片段，检出限为101 CFU/g，优化确定UvrD helicase、T4 gp32的终浓度分别为0.1 μg、5.0 μg，该方法用于检测猪肉、牛肉、羊肉、鸡肉中金黄色葡萄球菌的灵敏度高，耗时短，为猪肉、牛肉、羊肉、鸡肉中金黄色葡萄球菌的快速检测提供了新的方法，也为其它食品中金黄色葡萄球菌的快速检测奠定了基础。

Abstract:

A helicase-dependent isothermal DNA amplification (HAD) method is used for rapid and accurate detection Staphyloccocus aureus in pork, beef, mutton and chicken. A pair of oligonucleotide primers exclusively amplified the nuc gene of Staphyloccocus aureus, thus, Staphyloccocus aureus could be directly detected in pork, beef, mutton and chicken. The final concentrations of UvrD helicase and T4 gp32 were optimized. The amplification product was detected by electrophoresis. The results showed that HAD method had high specificity in detection of Staphyloccocus aureus in meat, but the other bacteria could not been amplificated. The amplification product had the same 216 bp length as the designed gene fragment, and the detection limit was 101 CFU/g. The optimized concentrations of UvrD helicase and T4 gp32 were 0.1 μg and 5.0 μg, respectively. HDA method was faster and more sensitive for detection of Staphyloccocus aureus in pork, beef, mutton and chicken, and established the foundation for the rapid detection of Staphylococcus aureus in other foods.